The present invention relates to pharmaceutical compositions comprising coagulation Factor VIII, said compositions being stabilized, without the addition of albumin, by divalent metal ions, in particular Zn2+. The invention also relates to a method for production of recombinant Factor VIII, comprising culturing said mammalian cells in medium free of plasma-derived protein and supplemented with divalent metal ions, in particular Zn2+.
Classic hemophilia or hemophilia A is the most common of the inherited bleeding disorders. It results from a chromosome X-linked deficiency of blood coagulation Factor VIII, and affects almost exclusively males with an incidence of between one and two individuals per 10,000. The X-chromosome defect is transmitted by female carriers who are not themselves hemophiliacs. The clinical manifestation of hemophilia A is an abnormal bleeding tendency and before treatment with Factor VIII concentrates was introduced, the mean life span for a person with severe hemophilia was less than 20 years.
The use of concentrates of Factor VIII from plasma has considerably improved the situation for the hemophilia patients. The mean life span has increased extensively, giving most of them the possibility to live a more or less normal life. However, there have been certain problems with the plasma derived concentrates and their use, the most serious of which have been the transmission of viruses. Although various virus inactivation methods have been developed, it appears likely that it will be impossible to render plasma-derived Factor VIII completely free from the risk of viral transmission.
The development of recombinant Factor VIII products, as opposed to plasma-derived Factor VIII, would apparently involve a lower risk for transmission of infectious agents. The molecular cloning of DNA coding for human Factor VIII was independently reported by research groups from Genentech Inc. (Gitschier, J. et al. (1984) Nature 312, 326-330; Wood, W. I. et al. (1984) Nature 312, 330-337; Vehar, G. A. et al. (1984) Nature 312, 337-342) and from Genetics Institute Inc. (Toole, J. J. et al. (1984) Nature 312, 342-347). Factor VIII mRNA encodes a precursor protein of 2351 amino acids including a 19 amino acid signal peptide; thus the mature Factor VIII protein is 2332 amino acids long. The amino acid sequence predicted a domain structure consisting of a triplicated A domain, a unique B domain and a duplicated C domain arranged in the order A1:A2: B:A3: C1: C2. During coagulation the B domain is removed by thrombin activation of the molecule and its function is unknown.
Characterization studies of recombinant human Factor VIII (Eaton, D. L. et al. (1987) J. Biol. Chem. 262, 3285-3290) showed that it is structurally and functionally very similar to plasma-derived Factor VIII. In plasma prepared in the presence of protease inhibitors, Factor VIII appeared as a complex of one heavy chain between 90-200 kDa (domains A1 and A2, with variable extensions of the B domain), in combination with one 80 kDa light chain (domains A3:C1:C2) (Andersson, L. O. et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2979-2983). The chains could be dissociated by EDTA, indicating that they are held together by metal ions. The C-terminal part of the heavy chain, containing the heavily glycosylated B-domain, is shown to be very sensitive to proteolytic attack by serine proteases.
Even in recombinant Factor VIII products there still remains a small but definite potential risk for transmission of infectious agents. Human albumin is a potential source of infection as it is used to stabilize the present recombinant Factor VIII products Kogenate(copyright) and Recombinate(copyright) (For a review, see Roddie, P. H. and Ludlam, C. A. (1997) Blood Reviews 11, 169-177). The presence of human parvovirus B19 DNA in recombinant, albumin containing Factor VIII products has been reported (Eis-Hxc3xcbinger, A. M. et al. (1996) Thrombosis and Haemostasis 76, p.1120).
A recombinant Factor VIII lacking the B domain, (r-VIII SQ), is produced by Pharmacia and Upjohn (for a review, see Berntorp, E. (1997) Thrombosis and Haemostasis 78, 256-260; see also EP-A-0506757). The r-VIII SQ protein, which consists of a 90 kDa heavy chain (domains A1:A2) and the 80 kDa light chain (domains A3:C1:C2), connected by a linker peptide, is produced in CHO cells cultured in medium which is serum-free but contains human serum albumin. Albumin is not required for stabilization of the final product, which instead contains Polysorbate 80, a non ionic detergent that has been shown to prevent activity losses caused by surface adsorption (cf. WO 94/07510).
A divalent metal ion is essential for the structural integrity and cofactor function of Factor VIII. However, little information is available regarding how metal ions fulfill these roles. Various models for the metal-dependent association of Factor VIII subunits have been proposed. Factor VIII has been suggested to circulate in normal plasma as a calcium-linked protein complex (Mikaelsson, M. et al. (1983) Blood 62, 1006-1015). Factor VIII activity has been reconstituted by recombining the subunits in the presence of Ca(II) or Mn(II) (Fay, P. J. (1988) Arch. Biochem. Biophys. 262, 525-531). Other authors have proposed that a copper atom is located between the A1 and A3 domains and is a structural prerequisite to maintain the association between the heavy and light chains (Bihoreau, N. et al. (1994) Eur. J. Biochem. 222, 41-48; Pan, Y. et al. (1995) Nature Structural Biology 2, 740-744; Pemberton, S. et al. (1997) Blood 89, 2413-2421).
U.S. Pat. No. 5,804,420 (Chan et al./Bayer Corporation) discloses a method for production of recombinant Factor VIII, comprising culturing host cells in medium free of plasma-derived protein and supplemented with polyols and copper ions.
By addition of Ca2+ ions and increase in the ionic strength when formulating r-VIII SQ as a solution it has been possible to reach a storage stability of a few months at +7xc2x0 C. (Fatourus, A. et al. (1997) Int. J. Pharm. 155, 121-131). Further improvement has been achieved by addition of large amounts of sucrose (Fatouros, A. et al. (1997) Pharm. Res. 14(12), 1679-1684). However, none of the formulations with a reasonably elevated osmolality had an acceptable long-term storage stability.
Replacement therapy with intravenous injection of Factor VIII is usually given in the patient""s home by a parent or the patient himself. Prior to administration the lyophilized Factor VIII concentrate has to be reconstituted under aseptic conditions, which is inconvenient and time-consuming. As a result there is often a substantial delay between the onset of bleeding symptoms and the treatment. This delay may increase the risk of chronic progressive joint damage.
A stable, ready-to-use solution of Factor VIII would be of great benefit to the patients. A convenient dosage form is expected to enhance therapy compliance, i.e. a more timely treatment, thereby reducing the likelihood of developing joint destruction. Besides the obvious advantages to the patients, elimination of the lyophilization step would simplify the manufacturing process and reduce both production and investment costs. At present there are no ready-to-use solutions of Factor VIII available. The stability of Factor VIII in solution is normally very poor; the same is true for B-domain deleted Factor VIII. Consequently, there is a need for a stable, ready-to-use composition of Factor VIII in order to enhance therapy compliance, and to simplify the manufacturing process.
Further, for plasma-derived Factor VIII, the current use of citrate anticoagulant reduces the levels of free divalent ions in the source plasma. For recombinant Factor VIII the yield in the cell culture process may be dependent on optimum concentrations of divalent metal ions in the cell culture medium. Thus there is also a need for improvements in the process of purification of Factor VIII, including the cell cultivation step in the production of recombinant Factor VIII.